Aldehyde Groups And The Schiff Reagent

The ability of bleached basic fuchsin (Schiff reagent) to react with aldehyde groups results in a distinctive red color and is the basis of the periodic acid-Schiff and Feulgen reactions

The periodic acid-Schiff (PAS) reaction stains carbohydrates and carbohydrate-rich macromolecules. It is used to demonstrate glycogen in cells, mucus in various cells and tissues, the basement membrane that underlies epithelia, and reticular fibers in connective tissue. The Feulgen reac tion, which employs a mild hydrochloric acid hydrolysis, is used to stain DNA.

The PAS reaction is based on the following facts:

• Hexose rings of carbohydrates contain adjacent carbons, each of which bears a hydroxyl (—OH) group.

• Hexosamines of glycosaminoglycans contain adjacent carbons, one of which bears an —OH group, while the other bears an amino (—NH2) group.

• Periodic acid cleaves the bond between these adjacent carbon atoms and forms aldehyde groups.

• These aldehyde groups react with the Schiff reagent to give a distinctive magenta color.

The PAS staining of basement membrane (Fig. 1.2) and reticular fibers is based on the content or association of proteoglycans (complex carbohydrates associated with a protein core). PAS staining is an alternative to silver impregnation methods, which are also based on reaction with the sugar molecules in the proteoglycans.

The Feulgen reaction is based on cleavage of purines from the deoxyribose of DNA by mild acid hydrolysis; the sugar ring then opens with the formation of aldehyde groups. Again, it is the newly formed aldehyde groups that react with the Schiff reagent to give the distinctive magenta color. The reaction of the Schiff reagent with DNA is stoichiometric and can be used, therefore, in spectrophoto-metric methods to quantify the amount of DNA in the nucleus of a cell. RNA does not stain with the Schiff reagent because it lacks deoxyribose.

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